Method for the production of 1,3-propanediol by recombinant organisms comprising genes for coenzyme B12 synthesis

ABSTRACT

Recombinant organisms are provided comprising genes encoding cob(II)alamin reductase, cob(I)alamin adenosyltransferase, glycerol dehydratase and 1,3-propanediol oxidoreductase activities useful for the production of 1,3-propanediol from a variety of carbon substrates.

This application is a divisional of U.S. patent application Ser. No.09/310,001 filed May 11, 1999, now U.S. Pat. No. 7,074,608, which claimsthe benefit of U.S. Provisional Application No. 60/085,214 filed May 12,1998, both of which are incorporated by reference herein in theirentireties.

FIELD OF INVENTION

The present invention relates to the field of molecular biology and theuse of recombinant organisms for the production of 1,3-propanediol. Morespecifically it describes the expression of cloned genes that affect thetransformation of coenzyme B₁₂ precursors to coenzyme B₁₂ in conjunctionwith genes that effectively convert a carbon substrate to1,3-propanediol.

BACKGROUND

1,3-Propanediol is a monomer useful in the production of polyesterfibers and the manufacture of polyurethanes and cyclic compounds.

A variety of chemical routes to 1,3-propanediol are known. For example,1,3-propanediol is prepared from 1) ethylene oxide over a catalyst inthe presence of phosphine, water, carbon monoxide, hydrogen and an acid;2) by the catalytic solution phase hydration of acrolein followed byreduction; or 3) from hydrocarbons such as glycerol, reacted in thepresence of carbon monoxide and hydrogen over catalysts having atomsfrom Group VIII of the periodic table. Although it is possible togenerate 1,3-propanediol by these chemical methods, they are expensiveand generate waste streams containing environmental pollutants.

It has been known for over a century that 1,3-propanediol can beproduced from the fermentation of glycerol. Bacterial strains able toproduce 1,3-propane-diol have been found, for example, in the groupsCitrobacter, Clostridium, Enterobacter, Ilyobacter, Klebsiella,Lactobacillus, and Pelobacter. In each case studied, glycerol isconverted to 1,3-propanediol in a two-step, enzyme-catalyzed reactionsequence. In the first step, a dehydratase catalyzes the conversion ofglycerol to 3-hydroxypropionaldehyde (3-HP) and water (Equation 1). Inthe second step, 3-HP is reduced to 1,3-propanediol by a NAD⁺-linkedoxidoreductase (Equation 2).Glycerol→3-HP+H₂O  (Equation 1)3-HP+NADH+H⁺→1,3-Propanediol+NAD⁺  (Equation 2)The 1,3-propanediol is not metabolized further and, as a result,accumulates in high concentration in the media. The overall reactionconsumes a reducing equivalent in the form of a cofactor, reducedβ-nicotinamide adenine dinucleotide (NADH), which is oxidized tonicotinamide adenine dinucleotide (NAD⁺).

The production of 1,3-propanediol from glycerol is generally performedunder anaerobic conditions using glycerol as the sole carbon source andin the absence of other exogenous reducing equivalent acceptors. Forexample, in strains of Citrobacter, Clostridium, and Klebsiella, aparallel pathway for glycerol operates under these conditions whichfirst involves oxidation of glycerol to dihydroxyacetone (DHA) by aNAD⁺- (or NADP⁺-) linked glycerol dehydrogenase (Equation 3). The DHA,following phosphorylation to dihydroxyacetone phosphate (DHAP) by a DHAkinase (Equation 4), becomes available for biosynthesis and forsupporting ATP generation via, for example, glycolysis.Glycerol+NAD⁺→DHA+NADH+H⁺  (Equation 3)DHA+ATP→DHAP+ADP  (Equation 4)In contrast to the 1,3-propanediol pathway, this pathway may providecarbon and energy to the cell and produces rather than consumes NADH.

In Klebsiella pneumoniae and Citrobacter freundii, the genes encodingthe functionally linked activities of glycerol dehydratase (dhaB),1,3-propanediol oxidoreductase (dhaT), glycerol dehydrogenase (dhaD),and dihydroxyacetone kinase (dhaK) are encompassed by the dha regulon.The dha regulons from Citrobacter and Klebsiella have been expressed inEscherichia coli and have been shown to convert glycerol to1,3-propanediol.

The biological production of 1,3-propanediol requires glycerol as asubstrate for a two-step sequential reaction in which a dehydrataseenzyme (typically a coenzyme B₁₂-dependent dehydratase) convertsglycerol to an intermediate, 3-hydroxypropionaldehyde, which is thenreduced to 1,3-propanediol by a NADH- (or NADPH) dependentoxidoreductase. The complexity of the cofactor requirements necessitatesthat a whole cell catalyst be used for an industrial processincorporating this reaction sequence for the production of1,3-propanediol. A process for the production of 1,3-propanediol fromglycerol using an organism containing a coenzyme B₁₂-dependent dioldehydratase is described in U.S. Pat. No. 5,633,362 (Nagarajan et al.).However, the process is not limited to the use of glycerol as feedstock.Glucose and other carbohydrates are suitable substrates and, recently,these substrates have been shown to be substrates for 1,3-propanediolproduction. Carbohydrates are converted to 1,3-propanediol using mixedmicrobial cultures where the carbohydrate is first fermented to glycerolby one microbial species and then converted to 1,3-propanediol by asecond microbial species U.S. Pat. No. 5,599,689 (Haynie et al.).However, a single organism able to convert carbohydrates to1,3-propanediol is preferred for reasons of simplicity and economy. Suchan organism is described in U.S. Pat. No. 5,686,276 (Laffend et al.);and in U.S. Ser. No. 60/030,601 and U.S. Ser. No. 08/969,683.

Glycerol dehydratase and diol dehydratase are coenzyme B₁₂-dependentenzymes which catalyze the conversion of glycerol to 3-HP (Toraya, T.,In Metalloenzymes Involving Amino Acid-Residue and Related Radicals;Sigel, H. and Sigel, A., Eds.; Metal Ions in Biological Systems; MarcelDekker: New York, 1994; Vol. 30, pp 217-254). Coenzyme B₁₂ may beprovided by the whole cell catalyst through de novo synthesis. However,if the coenzyme B₁₂ requirement of the B₁₂-dependent dehydratasesexceeds the de novo synthesis capacity of the whole cell catalyst or ifthe whole cell catalyst lacks the de novo synthesis capacity, thencoenzyme B₁₂ or coenzyme B₁₂ precursors may be provided in the reactionmedium. Due to the cost and instability of coenzyme B₁₂, mediumsupplementation with coenzyme B₁₂ precursors is preferred; and this thenrequires the conversion of these precursors to coenzyme B₁₂. Inaddition, glycerol dehydratase and diol dehydratase undergo inactivationwhich involves loss of the 5′-deoxyadenosyl moiety from coenzyme B₁₂ andthe formation of hydroxocobalamin and/or cob(II)alamin. (Toraya, T.,supra.) Thus, readenosylation of hydroxocobalamin and/or cob(II)alaminis required for the recycling of coenzyme B₁₂.

Vitamin B₁₂ (cyanocobalamin) and hydroxocobalamin are stable,commercially available coenzyme B₁₂ precursors which are readily takenup by microorganisms. Conversion of these precursors, both Co(III)species, to coenzyme B₁₂ (5′-deoxyadenosyl cobalamin) involves: 1.)reduction of Co(III) to Co(II) (i.e., formation of cob(II)alamin by aaquacobalamin reductase), 2.) reduction of Co(II) to Co(I) (i.e.,formation of cob(I)alamin by a cob(II)alamin reductase), and 3.)ATP-dependent adenosylation of cob(I)alamin by a cob(I)alaminadenosyltransferase to form coenzyme B₁₂ Enzymes associated with thesefunctions have been described for Salmonella typhimurium, Pseudomonasdenitrificans, and Clostridium tetanomorphum. Suh andEscalante-Semerena, J. Bacteriol. 177, 921-925 (1995) and referencestherein. Similar systems have been described for Euglena gracilis(Watanabe et al., Arch. Biochem. Biophys. 305, 421-427 (1993)),Chlamydomonas reinhardtii (Watanabe et al., Biochim. Biophys. Acta 1075,36-41 (1991)), and mammalian cells (Pezacka, E. H., Biochim. Biophys.Acta 1157, 167-177 (1993)).

The problem to be solved is how to biologically produce 1,3-propanediolby a single recombinant organism containing genes facilitating thesynthesis of B₁₂ coenzyme in the presence of a B₁₂-dependent dehydrataseenzyme.

SUMMARY OF THE INVENTION

Applicants have solved the stated problem. They provide a singleorganism capable of the dehydratase-mediated bioconversion of afermentable carbon source directly to 1,3-propanediol, where B₁₂coenzyme synthesis is effected by foreign genes encoding aquacobalaminreductase, cob(II)alamin reductase and cob(I)alamin adenosyltransferaseactivities. Glucose and glycerol are used as model substrates and thebioconversion is applicable to any existing microorganism.

The present invention provides a process for the production of1,3-propanediol from a transformed host cell comprising (i) contacting atransformed host cell with at least one fermentable carbon source and aneffective amount of coenzyme B₁₂ precursor whereby 1,3-propanediol isproduced; wherein said host cell comprises: a) at least one copy of agene encoding a protein having a dehydratase activity; b) at least onecopy of a gene encoding a protein having an oxidoreductase activity; c)at least one copy of a gene encoding a protein having a aquacobalaminreductase activity; d) at least one copy of a gene encoding a proteinhaving a cob(II)alamin reductase activity; and e) at least one copy of agene encoding a protein having a cob(I)alamin adenosyltransferaseactivity; wherein at least one of the genes of (c), (d) or (e) isintroduced into the host cell, and (ii) recovering the 1,3-propanediolproduced in (i). The dehydratase activity of (i)(a) may be from either aglycerol dehydratase enzyme or a diol dehydratase enzyme. The processmay be regulated by selectively inhibiting any one of the genes of(i)(c), (i)(d), or (i)(e) to alter the metabolism of coenzyme B₁₂precursor. The effective amount of coenzyme B₁₂ precursor is at a 0.1-to 10.0-fold molar ratio to the amount of dehydratase present, theimproved production measured against a bioprocess where the genes arenot present in multicopy.

The invention further provides a transformed host organism containing(a) at least one copy of a gene encoding a protein having a dehydrataseactivity; (b) at least one gene encoding a protein having anoxidoreductase activity; (c) at least one copy of a gene encoding aprotein having an aquacobalamin reductase activity; (d) at least onecopy of a gene encoding a protein having a cob(II)alamin reductaseactivity; (e) and at least one copy of a gene encoding a protein havinga cob(I)alamin adenosyltransferase activity, wherein at least one of thegenes of (i)(c), (i)(d), or (i)(e) is introduced into the host cell.

BRIEF DESCRIPTION OF SEQUENCE LISTING

Applicants have provided 25 sequences in conformity with Rules for theStandard Representation of Nucleotide and Amino Acid Sequences in PatentApplications (Annexes I and II to the Decision of the President of theEPO, published in Supplement No. 2 to OJ EPO, 12/1992), with 37 C.F.R.1.821-1.825 and Appendices A and B (Requirements for ApplicationDisclosures Containing Nucleotides and/or Amino Acid Sequences) withWorld Intellectual Property Organization (WIPO) Standard ST.25 (1998)and the sequence listing requirements of the EPO and PCT (Rules 5.2 and49.5(a-bis), and Section 208 and Annex C of the AdministrativeInstructions). The Sequence Descriptions contain the one letter code fornucleotide sequence characters and the three letter codes for aminoacids as defined in conformity with the IUPAC-IYUB standards describedin Nucleic Acids Research 13:3021-3030 (1985) and in the BiochemicalJournal 219 (No. 2):345-373 (1984) which are herein incorporated byreference.

SEQ ID NO:1 is the nucleotide sequence for btuR, encoding the E. colicob(I)alamin adenosyltransferase enzyme.

SEQ ID NO:2 is the nucleotide sequence for cobA, encoding the Salmonellatyphimurium cob(I)alamin adenosyltransferase enzyme.

SEQ ID NO:3 is the nucleotide sequence for cobO, encoding thePseudomonas denitrificans cob(I)alamin adenosyltransferase enzyme.

SEQ ID NO:4 is the nucleotide sequence for dhaB1, encoding the α subunitof the glycerol dehydratase enzyme.

SEQ ID NO:5 is the nucleotide sequence for dhaB2, encoding the β subunitof the glycerol dehydratase enzyme.

SEQ ID NO:6 is the nucleotide sequence for dhaB3, encoding the γ subunitof the glycerol dehydratase enzyme.

SEQ ID NO:7 the nucleotide sequence for dhaT, encoding Klebsiellaoxidoreductase enzyme.

SEQ ID NO:8 is a universal primer used in the isolation of theCob(II)alamin reductase gene.

SEQ ID NO:9 is the nucleotide sequence for the yciK gene islolated fromE. coli.

SEQ ID NO:10 is the nucleotide sequence for PHK28-26 a 12.1 kbEcoRI-SalI fragment containing the dha operon.

SEQ ID NO:11 is the nucleotide sequence for a multiple cloning site andterminator sequence used in the construction of the expression vectorpTacIQ.

SEQ ID NO:12-19 are primers used in the construction of expressionvectors of the present invention.

SEQ ID NO:20 is the nucleotide sequence for an insert in pCL1920, usedin the construction of the expression cassette for dhaT and dhaB(1,2,3).

SEQ ID NO:21 is the nucleotide sequence for the glucose isomerasepromoter sequence from Streptomyces.

SEQ ID NO:22-25 are primers used in the construction of expressionvectors of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for biologically producing1,3-propanediol from a fermentable carbon source in a single recombinantorganism. The method incorporates a microorganism containing genesencoding glycerol dehydratase, 1,3-propanediol oxidoreductase,aquacobalamin reductase, cob(II)alamin reductase, and cob(I)alaminadenosyltransferase. The recombinant microorganism is contacted with acarbon substrate (preferably glucose or glycerol) and 1,3-propanediol isisolated from the growth media.

The present method provides a rapid, inexpensive and environmentallyresponsible source of 1,3-propanediol monomer useful in the productionof polyesters and other polymers.

The following definitions are to be used to interpret the claims andspecification.

The term “aquacobalamin reductase” refers to an enzyme responsible forthe reduction of aquacobalamin to cob(II)alamin which involves thereduction of Co(III) to Co(II). Typical of aquacobalamin reductase is EC1.6.99.8.

The term “cob(II)alamin reductase” refers to an enzyme responsible forthe reduction of cob(II)alamin to cob(I)alamin which involves thereduction of Co(II) to Co(I). Typical of cob(II)alamin reductase is EC1.6.99.9. For purposes of the present invention, the terms“aquacobalamin reductase” and “cob(II)alamin reductase” include thosereductases which catalyze the corresponding reactions starting fromvitamin B₁₂.

The term “cob(I)alamin adenosyltransferase” refers to an enzymeresponsible for the transfer of a deoxyadenosyl moiety from ATP to thereduced corrinoid. Typical of cob(I)alamin adenosyltransferase is EC2.5.1.17. Cob(I)alamin adenosyltransferase is encoded by the gene “btuR”(GenBank M21528) (SEQ ID NO:1) in Escherichia coli, “cobA” (GenBankL08890) (SEQ ID NO:2) in Salmonella typhimurium, and “cobO” (SEQ ID NO3)(GenBank M62866) in Pseudomonas denitrificans.

The terms “coenzyme B₁₂” and “adenosylcobalamin” are usedinterchangeably to mean 5′-deoxyadenosylcobalamin. Hydroxocobalamin isthe derivative of coenzyme B₁₂ where the upper axial 5′-deoxyadenosylligand is replaced with a hydroxy moiety. Aquacobalamin is theunprotonated form of hydroxocobalamin. The terms “vitamin B₁₂” and“cyanocobalamin” are used interchangeably and refer to the derivative ofcoenzyme B₁₂ where the upper axial 5′-deoxy′5′-adenosyl ligand isreplaced with a cyano moiety. The term “coenzyme B₁₂ precursor” refersto a derivation of coenzyme B₁₂ where the upper axial 5′-deoxyadenosylligand is replaced. An “effective amount” of coenzyme B₁₂ precursor willmean that coenzyme B₁₂ precursor is present in the system atapproximately a 0.1- to 10.0-fold molar ratio to the amount ofdehydratase enzyme present.

The terms “glycerol dehydratase” or “dehydratase enzyme” refer to thepolypeptide(s) responsible for a coenzyme B₁₂-dependent enzyme activitythat is capable of isomerizing or converting a glycerol molecule to theproduct 3-hydroxypropionaldehyde. For the purposes of the presentinvention the dehydratase enzymes include a glycerol dehydratase(GenBank U09771, U30903) and a diol dehydratase (GenBank D45071) havingpreferred substrates of glycerol and 1,2-propanediol, respectively.Glycerol dehydratase of K pneumoniae ATCC 25955 is encoded by the genesdhaB1, dhaB2, and dhaB3 identified as SEQ ID NOS:4, 5, and 6respectively. The dhaB1, dhaB2 and dhaB3 genes code for the α, β, and γsubunits of the glycerol dehydratase enzyme, respectively. Glyceroldehydratase and diol dehydratase enzymes are complexes (with an α₂β₂γ₂subunit composition) that bind coenzyme B₁₂ with a 1:1 stoichiometry.

An “effective amount” of coenzyme B₁₂ precursor (or vitamin B₁₂) willmean that coenzyme B₁₂ precursor (or vitamin B₁₂) is present in thesystem at a molar ratio of between 0.1 and 10, relative to thedehydratase enzyme.

The terms “oxidoreductase” or “1,3-propanediol oxidoreductase” refer tothe polypeptide(s) responsible for an enzyme activity that is capable ofcatalyzing the reduction of 3-hydroxypropionaldehyde to 1,3-propanediol.1,3-Propanediol oxidoreductase includes, for example, the polypeptideencoded by the dhaT gene (GenBank U09771, U30903) and is identified asSEQ ID NO:7.

The terms “polypeptide” and “protein” are used interchangeably.

The terms “fermentable carbon substrate” and “fermentable carbon source”refer to a carbon source capable of being metabolized by host organismsof the present invention and particularly carbon sources selected fromthe group consisting of monosaccharides, oligosaccharides,polysaccharides, glycerol, dihydroxyacetone and one-carbon substrates ormixtures thereof.

The terms “host cell” or “host organism” refer to a microorganismcapable of receiving foreign or heterologous genes and of expressingthose genes to produce an active gene product.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

The terms “encoding” and “coding” refer to the process by which a gene,through the mechanisms of transcription and translation, produces anamino acid sequence. It is understood that the process of encoding aspecific amino acid sequence includes DNA sequences that may involvebase changes that do not cause a change in the encoded amino acid, orwhich involve base changes which may alter one or more amino acids, butdo not affect the functional properties of the protein encoded by theDNA sequence. It is therefore understood that the invention encompassesmore than the specific exemplary sequences. Modifications to thesequence, such as deletions, insertions, or substitutions in thesequence which produce silent changes that do not substantially affectthe functional properties of the resulting protein molecule are alsocontemplated. For example, alterations in the gene sequence whichreflect the degeneracy of the genetic code, or which result in theproduction of a chemically equivalent amino acid at a given site, arecontemplated. Thus, a codon for the amino acid alanine, a hydrophobicamino acid, may be substituted by a codon encoding another lesshydrophobic residue (such as glycine), or a more hydrophobic residue(such as valine, leucine, or isoleucine). Similarly, changes whichresult in substitution of one negatively charged residue for another(such as aspartic acid for glutamic acid), or one positively chargedresidue for another (such as lysine for arginine), can also be expectedto produce a biologically equivalent product. Nucleotide changes whichresult in alteration of the N-terminal and C-terminal portions of theprotein molecule would also not be expected to alter the activity of theprotein. In some cases, it may in fact be desirable to make mutants ofthe sequence in order to study the effect of alteration on thebiological activity of the protein. Each of the proposed modificationsis well within the routine skill in the art, as is determination ofretention of biological activity in the encoded products. Moreover, theskilled artisan recognizes that sequences encompassed by this inventionare also defined by their ability to hybridize, under stringentconditions (0.1×SSC, 0.1% SDS, 65° C.), with the sequences exemplifiedherein.

The term “substantially similar” refers to the relationship betweennucleic acid fragments wherein the second contains changes in one ormore nucleotide bases relative to the first resulting in substitution ofone or more amino acids, but with no affect on the functional propertiesof the protein encoded by the DNA sequence. “Substantially similar” alsorefers to the effect of modifications (such as deletion or insertion ofone or more nucleotide bases) to the nucleic acid fragment of theinstant invention that do not substantially affect the functionalproperties of the resulting transcript vis-à-vis the ability to mediatealteration of gene expression by antisense or co-suppression technologyor of alteration of the functional properties of the resulting proteinmolecule. It is therefore understood that the invention encompasses morethan the specific exemplary sequences.

For example, it is well-known that alterations in a gene which result inthe production of a chemically equivalent amino acid at a given site maynevertheless not effect the functional properties of the encodedprotein. Thus, a codon for the amino acid alanine, a hydrophobic aminoacid, may be substituted by a codon encoding another less hydrophobicresidue (such as glycine) or a more hydrophobic residue (such as valine,leucine, or isoleucine). Similarly, changes which result in substitutionof one negatively charged residue for another (such as aspartic acid forglutamic acid) or one positively charged residue for another (such aslysine for arginine) can also be expected to produce a functionallyequivalent product. Nucleotide changes which result in alteration of theN-terminal and C-terminal portions of the protein molecule would alsonot be expected to alter the activity of the protein. Each of theproposed modifications is well within the routine skill in the art, asis determination of retention of biological activity of the encodedproducts. Moreover, the skilled artisan recognizes that substantiallysimilar sequences encompassed by this invention are also defined bytheir ability to hybridize, under stringent conditions (0.1×SSC, 0.1%SDS, 65° C.), with the sequences exemplified herein. Preferredsubstantially similar nucleic acid fragments of the instant inventionare those nucleic acid fragments whose DNA sequences are at least 80%identical to the DNA sequence of the nucleic acid fragments reportedherein. More preferred nucleic acid fragments are at least 90% identicalto the identical to the DNA sequence of the nucleic acid fragmentsreported herein. Most preferred are nucleic acid fragments that are atleast 95% identical to the DNA sequence of the nucleic acid fragmentsreported herein.

A “substantial portion” of an amino acid or nucleotide sequencecomprises enough of the amino acid sequence of a polypeptide or thenucleotide sequence of a gene to putatively identify that polypeptide orgene, either by manual evaluation of the sequence by one skilled in theart, or by computer-automated sequence comparison and identificationusing algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul, S. F., et al., (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more nucleotides is necessary inorder to putatively identify a polypeptide or nucleic acid sequence ashomologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene specific oligonucleotide probes comprising20-30 contiguous nucleotides may be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides of 12-15 bases may be usedas amplification primers in PCR in order to obtain a particular nucleicacid fragment comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises enough of the sequence tospecifically identify and/or isolate a nucleic acid fragment comprisingthe sequence. The instant specification teaches partial or completeamino acid and nucleotide sequences encoding one or more particularreductase proteins. The skilled artisan, having the benefit of thesequences as reported herein, may now use all or a substantial portionof the disclosed sequences for purposes known to those skilled in thisart. Accordingly, the instant invention comprises the complete sequencesas reported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

The term “expression” refers to the transcription and translation togene product from a gene coding for the sequence of the gene product.

The terms “plasmid”, “vector”, and “cassette” refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

The terms “transformation” and “transfection” refer to the acquisitionof new genes in a cell after the incorporation of nucleic acid. Theacquired genes may be integrated into chromosomal DNA or introduced asextrachromosomal replicating sequences. The term “transformant” refersto the product of a transformation.

The term “genetically altered” refers to the process of changinghereditary material by transformation or mutation.

The term “regulate” refers to control of the production of1,3-propanediol by selective inhibition of the genes encoding a proteinhaving an aquacobalamin reductase activity, of the genes encoding aprotein having a cob(II)alamin reductase activity, or of the genesencoding a protein having a cob(I)alamin adenosyltransferase activity.

The present invention involves the construction of a production organismthat incorporates the genetic machinery necessary to convert afermentable carbon substrate to 1,3-propanediol, in conjunction withgenes encoding enzymes needed for the biotransformation of coenzyme B₁₂precursor to coenzyme B₁₂. The genes involved in 1,3-propanediolproduction will include a dehydratase gene (typically a glycerol or dioldehydratase) and an oxidoreductase as well as other proteins expected toaid in the assembly or in maintaining the stability of the dehydrataseenzyme. These genes may be transgenes and introduced into the host cell,or may be endogenous. Genes responsible for the conversion of coenzymeB₁₂ precursor to coenzyme B₁₂ will include at least one copy of a geneencoding a protein having a aquacobalamin reductase activity; at leastone copy of a gene encoding a protein having a cob(II)alamin reductaseactivity, and at least one copy of a gene encoding a protein having acob(I)alamin adenosyltransferase activity. At least one of these geneswill be a transgene and introduced into the production cell. Thetransformed production cell is then grown under appropriate conditionsfor the production of 1,3-propanediol.

Recombinant organisms containing the necessary genes that will encodethe enzymatic pathway for the conversion of a carbon substrate to1,3-propanediol may be constructed using techniques well known in theart. In the present invention genes encoding glycerol dehydratase (dhaB)and 1,3-propanediol oxidoreductase (dhaT) were isolated from a nativehost such as Klebsiella and together with genes encoding aquacobalaminreductase, cob(II)alamin reductase and cob(I) alamin adenosyltransferase(btuR or cobA or cobO) isolated from native hosts such as E. coli, S.typhimurium or P. denitrificans) are used to transform host strains suchas E. coli strain DH5α or FM5; K pneumoniae strain ATCC 25955; K.oxytoca strain ATCC 8724 or M5a1, S. cerevisiae strain YPH499, P.pastoris strain GTS115, or A. niger strain FS1.

Rational for Using dhaB dhaT

Producing 1,3-propanediol from glucose can be accomplished by thefollowing series of steps. This series is representative of a number ofpathways known to those skilled in the art. Glucose is converted in aseries of steps by enzymes of the glycolytic pathway to dihydroxyacetonephosphate (DHAP) and 3-phosphoglyceraldehyde (3-PG). Glycerol is thenformed by either hydrolysis of DHAP to dihydroxyacetone (DHA) followedby reduction, or by reduction of DHAP to glycerol 3-phosphate (G3P)followed by hydrolysis. The hydrolysis step can be catalyzed by anynumber of cellular phosphatases which are known to be non-specific withrespect to their substrates or the activity can be introduced into thehost by recombination. The reduction step can be catalyzed by a NAD⁺ (orNADP⁺) linked host enzyme or the activity can be introduced into thehost by recombination. It is notable that the dha regulon contains aglycerol dehydrogenase (E.C. 1.1.1.6) which catalyzes the reversiblereaction of Equation 7.Glycerol→3-HP+H₂O  (Equation 5)3-HP+NADH+H⁺→1,3-Propanediol+NAD⁺  (Equation 6)Glycerol+NAD⁺→DHA+NADH+H⁺  (Equation 7)Glycerol is converted to 1,3-propanediol via the intermediate3-hydroxy-propionaldehye (3-HP) as has been described in detail above.The intermediate 3-HP is produced from glycerol, Equation 5, by adehydratase enzyme which can be encoded by the host or can introducedinto the host by recombination. This dehydratase can be glyceroldehydratase (E.C. 4.2.1.30), diol dehydratase (E.C. 4.2.1.28) or anyother enzyme able to catalyze this transformation. Glycerol dehydratase,but not diol dehydratase, is encoded by the dha regulon. 1,3-Propanediolis produced from 3-HP, Equation 6, by a NAD⁺- (or NADP⁺) linked hostenzyme or the activity can introduced into the host by recombination.

This final reaction in the production of 1,3-propanediol can becatalyzed by 1,3-propanediol dehydrogenase (E.C. 1.1.1.202) or otheralcohol dehydrogenases.

The dha regulon is comprised of several functional elements includingdhaK encoding a dihydroxyacetone kinase, dhaD encoding a glyceroldehydrogenase, dhaR encoding a regulatory protein, dhaT encoding a1,3-propanediol oxidoreductase as well as dhaB1, dhaB2, and dhaB3encoding the alpha, beta and gamma subunits of a glycerol dehydratase,respectively. Additionally, gene products designated as protein X,protein 1, protein 2, and protein 3 (corresponding to dhaBX, orfY, orfX,and orfW, respectively) are encoded within the dha regulon. While theprecise functions of these gene products are not well characterized, thegenes are linked to glycerol dehydratase (dhaB) or 1,3-propanedioloxidoreductase (dhaT) and are known to be useful for the production of1,3-propanediol. Coenzyme B₁₂ that is bound to glycerol dehydrataseoccasionally undergoes irreversible cleavage to form an inactivemodified coenzyme which is tightly bound to the dehydratase.Reactivation of the enzyme occurs by exchange of the bound, modifiedcoenzyme with free, intact coenzyme B₁₂. Protein X and at least oneother of protein 1, protein 2, and protein 3 are involved in theexchange process. (see U.S. Ser. No. 08/969,683). In the separate dioldehydratase system, genes designated as ddrA and ddrB, corresponding tothe genes encoding protein X and protein 2, respectively, are describedto be involved in the exchange process (Mori et al., J. Biol. Chem. 272,32034-32041 (1997)).

Glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase may beparticularly effective in the conversion of glucose to glycerol,required for the production of 1,3-propanediol (U.S. Ser. No.60/030,602). The term “glycerol-3-phosphate dehydrogenase” refers to apolypeptide responsible for an enzyme activity that catalyzes theconversion of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate(G3P). In vivo G3PDH may be NADH-, NADPH-, or FAD-dependent. TheNADH-dependent enzyme (EC 1.1.1.8) is encoded, for example, by severalgenes including GPD1 (GenBank Z74071×2), or GPD2 (GenBank Z35169×1), orGPD3 (GenBank G984182), or DAR1 (GenBank Z7407 1×2). The NADPH-dependentenzyme (EC 1.1.1.94) is encoded by gpsA (GenBank U321643, (cds197911-196892) G466746 and L45246). The FAD-dependent enzyme (EC1.1.99.5) is encoded by GUT2 (GenBank Z47047×23), or glpD (GenBankG147838), or glpABC (GenBank M20938). The term “glycerol-3-phosphatase”refers to a polypeptide responsible for an enzyme activity thatcatalyzes the conversion of glycerol-3-phosphate and water to glyceroland inorganic phosphate. Glycerol-3-phosphatase is encoded, for example,by GPP1 (GenBank Z47047×125), or GPP2 (GenBank U18813×11).

Gene Isolation

Methods of obtaining desired genes from a bacterial genome are commonand well known in the art of molecular biology. For example, if thesequence of the gene is known, suitable genomic libraries may be createdby restriction endonuclease digestion and may be screened with probescomplementary to the desired gene sequence. Once the sequence isisolated, the DNA may be amplified using standard primer directedamplification methods such as polymerase chain reaction (PCR) (U.S. Pat.No. 4,683,202) to obtain amounts of DNA suitable for transformationusing appropriate vectors.

Alternatively, cosmid libraries may be created where large segments ofgenomic DNA (35-45 kb) may be packaged into vectors and used totransform appropriate hosts. Cosmid vectors are unique in being able toaccommodate large quantities of DNA. Generally, cosmid vectors have atleast one copy of the cos DNA sequence which is needed for packaging andsubsequent circularization of the foreign DNA. In addition to the cossequence these vectors will also contain an origin of replication suchas ColE1 and drug resistance markers such as a gene resistant toampicillin or neomycin. Methods of using cosmid vectors for thetransformation of suitable bacterial hosts are well described inSambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989).

Typically to clone cosmids, foreign DNA is isolated and ligated, usingthe appropriate restriction endonucleases, adjacent to the cos region ofthe cosmid vector. Cosmid vectors containing the linearized foreign DNAare then reacted with a DNA packaging vehicle such as bacteriophage λ.During the packaging process the cos sites are cleaved and the foreignDNA are packaged into the head portion of the bacterial viral particle.These particles are then used to transfect suitable host cells such asE. coli. Once injected into the cell, the foreign DNA circularizes underthe influence of the cos sticky ends. In this manner large segments offoreign DNA can be introduced and expressed in recombinant host cells.

Isolation and Cloning of Genes Encoding Glycerol Dehydratase (dhaB) and1,3-propanediol Oxidoreductase (dhaT)

Identification and isolation of dhaB and dhaT were done essentially asdescribed in U.S. Pat. No. 5,686,276 and those methods are herebyincorporated by reference. Cosmid vectors and cosmid transformationmethods were used within the context of the present invention to clonelarge segments of genomic DNA from bacterial genera known to possessgenes capable of processing glycerol to 1,3-propanediol. Two1,3-propanediol positive transformants were analyzed and DNA sequencingrevealed extensive homology to the glycerol dehydratase gene (dhaB) fromC. freundii, demonstrating that these transformants contained DNAencoding the glycerol dehydratase gene. dhaB and dhaT were isolated andcloned into appropriate expression cassettes for co-expression inrecombinant hosts with genes encoding B₁₂ coenzyme synthesis.

Although the instant invention uses the isolated genes from within aKlebsiella cosmid, alternate sources of dehydratase genes include, butare not limited to, Citrobacter, Clostridia, Enterobacter, andSalmonella.

B₁₂ Coenzyme Genes

Rational for B₁₂ Coenzyme Genes

Adenosylcobalamin (coenzyme B₁₂) is an essential cofactor for glyceroldehydratase activity. The coenzyme is the most complex non-polymericnatural product known, and its synthesis in vivo is directed using theproducts of about 20-30 genes. Synthesis of coenzyme B₁₂ is found inprokaryotes, some of which are able to synthesize the compound de novo,while others can perform partial reactions. E. coli, for example, cannotfabricate the corrin ring structure, but is able to catalyze theconversion of cobinamide to corrinoid and can introduce the5′-deoxyadenosyl group.

Eukaryotes are unable to synthesize coenzyme B₁₂ de novo and insteadtransport vitamin B₁₂ and other coenzyme B₁₂ precursors from theextracellular milieu with subsequent conversion of the compound to itsfunctional form of the compound by cellular enzymes. Three enzymeactivities have been described for this series of reactions: 1)aquacobalamin reductase (EC 1.6.99.8) reduces Co(III) to Co(II); 2)cob(II)alamin reductase (EC 1.6.99.9) reduces Co(II) to Co(III); and 3)cob(I)alamin adenosyltransferase (EC 2.5.1.17) transfers a5′-deoxyadenosine moiety from ATP to the reduced corrinoid. This lastenzyme activity is the best characterized of the three and is encoded bycobA in S. typhimurium, btuR in E. coli and cobO in P. denitrificans.These three cob(I)alamin adenosyltransferase genes have been cloned andsequenced. Cob(I)alamin adenosyltransferase activity has been detectedin human fibroblasts and in isolated rat mitochondria (Fenton et al.,Biochem. Biophys. Res. Commun. 98, 283-9, (1981)). The two enzymesinvolved in cobalt reduction are poorly characterized and gene sequencesare not available. There are reports of an aquacobalamin reductase fromEuglena gracilis (Watanabe et al., Arch. Biochem. Biophys. 305, 421-7,(1993)) and a microsomal cob(III)alamin reductase is present in themicrosomal and mitochondrial inner membrane fractions from ratfibroblasts (Pezacka, Biochim. Biophys. Acta, 1157, 167-77, (1993)).

Supplementing culture media with vitamin B₁₂ may satisfy the need toproduce coenzyme B₁₂ for glycerol dehydratase activity in manymicroorganisms, but in some cases additional catalytic activities mayhave to be added or increased in vivo particularly when high levels of1,3-propanediol are desired. Enhanced synthesis of coenzyme B₁₂ ineukaryotes may be particularly desirable. Given the published sequencesfor genes encoding cob(I)alamin adenosyltransferase, the cloning andexpression of this gene could be accomplished by one skilled in the art.For example, it is contemplated that yeast, such as Saccharomyces, couldbe constructed so as to contain genes encoding cob(I)alaminadenosyltransferase in addition to the genes necessary to effectconversion of a carbon substrate such as glucose to 1,3-propanediol.Cloning and expression of the genes for cobalt reduction requires adifferent approach. This could be based on a selection in E. coli forgrowth on ethanolamine as sole C or N₂ source. In the presence ofcoenzyme B₁₂, ethanolamine ammonia-lyase enables growth of cells in theabsence of other C or N₂ sources. If E. coli cells contain a cloned genefor cob(I)alamin adenosyltransferase and random cloned DNA from anotherorganism, growth on ethanolamine in the presence of aquacobalamin shouldbe enhanced and selected for if the random cloned DNA encodes cobaltreduction properties to facilitate adenosylation of aquacobalamin.

Another approach to identifying and cloning the gene(s) for cobaltreduction is based on coenzyme B₁₂ repression of btuB expression. WhenE. coli is grown in the presence of coenzyme B₁₂ the expression of btuBis reduced, and if a btuB::lacZ fusion is constructed this repressioncan be observed as a reduction in β-galactosidase activity. Vitamin B₁₂will also repress expression of btuB::lacZ once it has beenadenosylated, but factors that prevent conversion to coenzyme B₁₂, suchas defects in cobalt reduction or adenosylation, lead to constitutiveexpression of btuB::lacZ. To identify cobalt reduction genes requiresthe initial selection or identification of Lac⁺ cells from a btuB::lacZstrain that has been subjected to mutagenesis, followed by positiveselection for growth on lactose, or by identifying Lac⁺ colonies onindicator plates. False positives due to btuR mutations could beminimized by having btuR present on a plasmid. A strain defective incobalt reduction can be transformed with cloned DNA from the same oranother species, and cloned cobalt reduction genes identified because ofa Lac⁻ phenotype resulting from coenzyme B₁₂ repression of btuR::lacZexpression.

Isolation of Genes Encoding Cob(I)alamin Adenosyltransferase

The genes encoding cob(I)alamin adenosyltransferase were cloned from twospecies of bacteria, btuR from E. coli strain DH5α(deoR endA1 gyrA96hsdR17(rk− mk+) recA1 relA1 supE44 thi-1 Δ(lacZYA-argFV169)) and cobAfrom S. typhimurium strain ATCC 23564. Primers were designed using thepublished sequence of btuR (Lundrigan and Kadner, J. Bact. 171, 154-161(1989)) such that PCR-amplification of the gene from E. coli could beachieved to give the complete coding sequence flanked by HindIII andBamHI sites to the 5′ end, and a PstI site to the 3′ end. A ribosomebinding site was present between the HindIII and BamHI sites at the 5′end to ensure adequate translation. The PCR product was cloned into theSrfI site of pCR-Script to give plasmid pAH61. A correctly constructedclone was confirmed by DNA analysis and by functional expression tocomplement an E. coli btuR mutant strain for 1,3-propanediol production.

Primers were also designed for the S. typhimurium cobA gene usingpublished sequence (Suh and Escalante-Semerena, Gene 129, 93-97 (1993))such that PCR-amplification of the gene could be achieved to give thecomplete coding sequence flanked by HindIII and BamHI sites to the 5′end, and a PstI site to the 3′ end. A ribosome binding site was presentbetween the HindIII and BamHI sites at the 5′ end to ensure adequatetranslation. The PCR product was cloned into the SrfI site of pCR-Scriptto give plasmid pAH63. A correctly constructed clone was confirmed byDNA analysis and by functional expression to complement an E. coli btuRmutant strain for 1,3-propanediol production.

Isolation of the Cob(II)Alamin Reductase Gene

Cob(II)alamin reductase has been purified 6300-fold to homogeneity fromP. denitrificans strain SC510 (Blanche et al., J. Bact. 174, 7452-7454(1992)). The N-terminal amino acid sequence was determined to be Met GluLys Thr Arg Leu, from which one skilled in the art can design a suitablepopulation of primers that encompasses all possible nucleotidevariations that encode this peptide [ATG GAR AAR ACS CGI CTI, whereR=A+G; S═C+G; I=Inosine] (SEQ ID NO: 8). The pool of primers thusobtained is used for PCR-amplification of the gene for cob(II)alaminreductase using either of two techniques. In one approach, chromosomalDNA from P. denitrificans is subjected to PCR with the pool of primersencoding Met Glu Lys Thr Arg Leu, together with random primers to effectsecond strand synthesis. PCR products are cloned into a plasmid, such aspCR-Script, and the cloned fragments screened by DNA sequence analysis.

Another approach is to first clone DNA from P. denitrificans into aplasmid such as pCR-Script, followed by PCR amplification using the poolof primers encoding Met Glu Lys Thr Arg Leu for first strand synthesis.Second strand synthesis is accomplished by using as a primer a sequencederived from the known plasmid sequence. The isolated complete orpartial sequence for cob(II)alamin reductase from P. denitrificans isused as a probe to identify and clone similar genes from other speciesthat encode this enzyme.

Development of an appropriate selection strategy based oncomplementation allows identification and isolation of the gene forcob(II)alamin reductase. Lundrigan and Kadner (J. Bact 171, 154-161(1989)) describe how btuR (adenosyltransferase) mutants influence btuB(outer-membrane B₁₂ binding protein) gene regulation. The btuR mutantsare identified because they do not repress btuB expression. This is doneby first making a gene fusion between btuB and lacZ. Growth of thesecells in the absence of vitamin B₁₂ or coenzyme B₁₂ leads toconstitutive expression of btuB::lacZ (so that B₁₂ receptors are presenton the cell surface) to give a Lac⁺phenotype. In wild type cells vitaminB₁₂ undergoes cobalt reduction, and is then converted to coenzyme B₁₂ bycob(I)alamin adenosyltransferase, and the resulting coenzyme B₁₂ causesrepression of btuB::lacZ to give a Lac⁻ phenotype. In btuR mutants thevitamin B₁₂ is not converted to coenzyme B₁₂, repression of btuB::lacZdoes not occur and a Lac⁺phenotype is observed on media containingvitamin B₁₂. Therefore, to isolate btuR mutants requires selection oridentification of Lac⁺ cells from a btuB::lacZ strain in the presence ofvitamin B₁₂. Since the cob(II)alamin reductase, like BtuR, functionsduring the conversion of vitamin B₁₂ to coenzyme B₁₂, the samerequirement for growth on media containing lactose and vitamin B₁₂ of abtuB::lacZ strain enables a positive selection for mutations incob(II)alamin reductase. Alternatively, such mutations are observed asLac⁺ colonies on indicator plates. False positives due to btuR mutationsare minimized by having btuR present on a multicopy plasmid. Isolationof mutations in the gene for cob(II)alamin reductase is achieved bychemical mutagenesis, UV light or by the use of transposons such as Tn5or Tn10. Strains with mutations in cob(II)alamin reductase arecomplemented using a cloned genomic library to give a Lac⁻ phenotype onLac indicator plates, leading to identification of the specific gene. Inaddition to using a library of cloned DNA to identify a cob(U)alaminreductase through complementation, defined fragments of cloned DNAencoding reductases are used to assess complementation. A fragment ofchromosomal DNA from E. coli (bearing the yciK gene (GenBank CO06550)(SEQ ID NO:9) encoding a dehydrogenase/reductase or a related sequencefrom other prokaryotes) is tested in the complementation assay for aLac⁻ phenotype resulting from reduction and adenosylation of vitamin B₁₂to form coenzyme B₁₂, which in turn will repress expression ofbtuB::lacZ. yciK, located immediately upstream of btuR, is transcribedin the same direction, and the termination codon (UGA) of yciK overlapswith the initiation codon (AUG) of btuR in the genomic sequence ATGA.This sequential arrangement of termination and initiation codons is acharacteristic of genes that are translationally coupled andco-regulated (Gatenby et al., Proc. Natl. Acad. Sci. USA 86, 4066-4070(1989)). E. coli yciK is a particularly preferred gene to enable cobaltreduction during the synthesis of coenzyme B₁₂.

The skilled artisan will appreciate that utility of thedehydrogenase/reductase activity encoded by yciK will not be limited tothis specific gene or enzyme but will include homologues of the gene orenzyme including genes and enzymes that are substantially similar to thegene or enzyme and those genes having about 80% identity to the gene,where those having 90% identity re preferred, and where that havingabout 95% identity are most preferred.

In addition to selections based on a Lac phenotype, it is possible touse E. coli strains which carry a defective metE gene that encodes acobalamin-independent methionine synthase, but which retain a functionalmetH gene encoding a cobalamin-dependent methionine synthase. An exampleof such a strain is CAG18491(F⁻, λ⁻, rph-1, metE3079::Tn10), amethionine auxotroph unless vitamin or coenzyme B₁₂ is added to themedia. Mutagenesis of CAG18491 and growth on minimal media containingcoenzyme B₁₂, followed by colony testing on minimal media containingvitamin B₁₂, allows identification of cells that have lost the abilityto convert vitamin to coenzyme B₁₂, but which can still use coenzyme B₁₂during the synthesis of methionine by the MetH methionine synthase. Thecells identified in this screen are defective in one of the two cobaltreduction steps, or in adenosylation of vitamin B₁₂. Introduction of acloned btuR plasmid into cells that are methionine auxotrophs on vitaminB₁₂ but are prototrophs on coenzyme B₁₂ will identify cells that remainMet⁻ on vitamin B₁₂, even though btuR is present. Alternatively, aplasmid bearing btuR is added to the metE strain prior to mutagenesisand selection. Cells that are defective in one or both of the two cobaltreduction steps can be used to screen a genomic library for clones thatrestore prototrophic growth on minimal media with vitamin B₁₂. Topreclude cloning of the metE gene in this selection it is important toprepare the genomic library from a strain that has a defective metEgene. Plasmids obtained from this selection will encode an enzymecapable of reducing cobalt. Assaying for cob(II)alamine reductaseconfirms this property.

Isolation of the Aquacobalamin Reductase Gene

Aquacobalamin reductase is purified from, but is not limited to,Pseudomonas, Escherichia, Salmonella, Klebsiella or Citrobacter, asdescribed by Watanabe and Nakono (Methods Enzymol. 281, 289-305 (1997))or with variations thereof. An enzyme assay is used that measures thedecrease in absorbance of aquacobalamin at 525 nm (Watanabe et al., J.Nutr. 126, 2947-2951 (1996)). The N-terminal amino acid sequence of theprotein is determined, from which one skilled in the art can design acollection of primers that includes all possible nucleotide variationsthat encode the N-terminal peptide. The pool of primers thus obtained isused for PCR-amplification of the gene for aquacobalamin reductase usingeither of two techniques. In one approach, chromosomal DNA is subjectedto PCR with the pool of primers encoding the N-terminal peptide,together with random primers to effect second strand synthesis. PCRproducts are cloned into a plasmid, such as pCR-Script, and the clonedfragments screened by DNA sequence analysis. Another approach is tofirst clone chromosomal DNA into a plasmid such as pCR-Script, followedby PCR amplification using the pool of primers encoding the N-terminalpeptide for first strand synthesis. Second strand synthesis isaccomplished by using as a primer a sequence derived from the knownplasmid sequence. The isolated complete or partial sequence foraquacobalamin reductase is used as a probe to identify and clone similargenes from other species that encode this enzyme.

Identification and isolation of the gene for aquacobalamin reductasearises from an appropriate selection strategy based on complementation.Lundrigan and Kadner (J. Bact 171, 154-161 (1989)) describe how btuR(adenosyltransferase) mutants influence btuB (outer-membrane B₁₂ bindingprotein) gene regulation. The btuR mutants are identified because theydo not repress btuB expression. This is done by first making a genefusion between btuB and lacZ. Growth of these cells in the absence ofvitamin B₁₂ or coenzyme B₁₂ leads to constitutive expression ofbtuB::lacZ (so that B₁₂ receptors are present on the cell surface) togive a Lac⁺ phenotype. In wild type cells vitamin B₁₂ undergoes cobaltreduction, and is then converted to coenzyme B₁₂ by cob(I)alaminadenosyltransferase. The resulting coenzyme B₁₂ causes repression ofbtuB::lacZ to give a Lac⁻ phenotype. In btuR mutants the vitamin B₁₂ isnot converted to coenzyme B₁₂, repression of btuB::lacZ does not occur,and a Lac⁺ phenotype is observed on media containing vitamin B₁₂.Therefore, to isolate btuR mutants requires selection or identificationof Lac⁺ cells from a btuB::lacZ strain in the presence of vitamin B₁₂.Since the aquacobalamin reductase, like BtuR, functions during theconversion of vitamin B₁₂ to coenzyme B₁₂, the same requirement forgrowth on media containing lactose and vitamin B₁₂ of a btuB::lacZstrain enables a positive selection for mutations in aquacobalaminreductase. Alternatively, such mutations are observed as Lac⁺ colonieson indicator plates. False positives due to btuR mutations are minimizedby having btuR present on a multicopy plasmid. Isolation of mutations inthe gene for aquacobalamin reductase is achieved by chemicalmutagenesis, UV light or by the use of transposons such as Tn5 or Tn10.Strains with mutations in aquacobalamin reductase are complemented usinga cloned genomic library to give Lac⁻ phenotype on Lac indicator plates,leading to identification of the specific gene. In addition to suchcomplementation using a library of cloned DNA to identify anaquacobalamin reductase, defined fragments of cloned DNA encoding areductase are used to assess complementation

In addition to selections based on a Lac phenotype, it is possible touse E. coli strains which carry a defective metE gene that encodes acobalamin-independent methionine synthase, but which retain a functionalmetH gene encoding a cobalamin-dependent methionine synthase. Theprocedure follows that described above for the isolation ofCob(II)alamin reductase gene. Plasmids obtained from this selection willencode an enzyme capable of reducing cobalt. Assaying for aquacobalaminreductase confirms this property.

Host Cells

Suitable host cells for the recombinant production 1,3-propanediol bythe coexpression of a gene encoding a dehydratase enzyme and the genesencoding cob(I)alamin adenosyltransferase, aquacobalamin reductase andcob(II)alamin reductase may be either prokaryotic or eukaryotic and willbe limited only by their ability to express active enzymes. Preferredhosts will be those typically useful for production of 1,3-propanediolor glycerol such as Citrobacter, Enterobacter, Clostridium, Klebsiella,Aerobacter, Lactobacillus, Aspergillus, Saccharomyces,Schizosaccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida,Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia,Salmonella, Bacillus, Streptomyces and Pseudomonas. Most preferred inthe present invention are E. coli, Klebsiella species and Saccharomycesspecies.

E. coli and Klebsiella species are particularly preferred hosts. Strainsof Klebsiella pneumoniae are known to produce 1,3-propanediol when grownon glycerol as the sole carbon. It is contemplated that Klebsiella canbe genetically altered to produce 1,3-propanediol from monosaccharides,oligosaccharides, polysaccharides, or one-carbon substrates.

Vectors And Expression Cassettes

The present invention provides a variety of vectors and transformationand expression cassettes suitable for the cloning, transformation andexpression of genes encoding a suitable dehydratase and of geneseffecting the conversion of coenzyme B₁₂ precursors to coenzyme B₁₂ intoa suitable host cell. Suitable vectors will be those which arecompatible with the bacterium used as a host cell. Suitable vectors canbe derived, for example, from a bacteria, a virus (such as bacteriophageT7 or a M-13 derived phage), a cosmid, a yeast or a plant. Protocols forobtaining and using such vectors are known to those in the art.(Sambrook et al., Molecular Cloning: A Laboratory Manual—volumes 1,2,3(Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989)).

Typically, the vector or cassette contains sequences directingtranscription and translation of the relevant gene, a selectable marker,and sequences allowing autonomous replication or chromosomalintegration. Suitable vectors comprise a region 5′ of the gene whichharbors transcriptional initiation controls and a region 3′ of the DNAfragment which controls transcriptional termination. It is mostpreferred when both control regions are derived from genes homologous tothe transformed host cell although it is to be understood that suchcontrol regions need not be derived from the genes native to thespecific species chosen as a production host.

Initiation control regions or promoters, useful to drive expression ofthe relevant genes of the present invention in the desired host cell,are numerous and familiar to those skilled in the art. Virtually anypromoter capable of driving these genes is suitable for the presentinvention including but not limited to CYC1, HIS3, GAL1, GAL10, ADH1,PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful forexpression in Saccharomyces); AOX1 (useful for expression in Pichia);and lac, trp, λP_(L), λP_(R), T7, tac, and trc (useful for expression inE. coli).

Termination control regions may also be derived from various genesnative to the preferred hosts. Optionally, a termination site may beunnecessary; however, it is most preferred if included.

For effective expression of the instant enzymes, DNA encoding theenzymes are linked operably through initiation codons to selectedexpression control regions such that expression results in the formationof the appropriate messenger RNA.

Transformation of Suitable Hosts and Expression of Genes for theProduction of 1,3-propanediol

Once suitable cassettes are constructed they are used to transformappropriate host cells. Introducing into the host cell the cassettecontaining the genes encoding cob(I)alamin adenosyltransferase,aquacobalamin reductase, cob(II)alamin reductase, glycerol dehydratase(dhaB), and 1,3-propanediol oxidoreductase (dhaT) (either separately ortogether) may be accomplished by known procedures includingtransformation (e.g., using calcium-permeabilized cells,electroporation) or by transfection using a recombinant phage virus.(Sambrook et al., supra.)

In the present invention, E. coli FM5 containing the genes encodingglycerol dehydratase (dhaB), 1,3-propanediol oxidoreductase (dhaT),aquacobalamin reductase, cob(II)alamin reductase, and cob(I)alaminadenosyltransferase is used to convert vitamin B₁₂ supplied in the mediato coenzyme B₁₂ to enable glycerol dehydratase to function.

Media and Carbon Substrates

Fermentation media in the present invention must contain suitable carbonsubstrates. Suitable substrates may include but are not limited toglycerol, dihydroxyacetone, monosaccharides such as glucose andfructose, oligosaccharides such as lactose or sucrose, polysaccharides(such as starch or cellulose), or mixtures thereof, and unpurifiedmixtures from renewable feedstocks (such as cheese whey permeate,cornsteep liquor, sugar beet molasses, and barley malt). Additionally,the carbon substrate may also be one-carbon substrates (such as carbondioxide or methanol) for which metabolic conversion into key biochemicalintermediates has been demonstrated.

Glycerol production from single carbon sources (e.g., methanol,formaldehyde, or formate) has been reported in methylotrophic yeasts(Yamada et al., Agric. Biol. Chem., 53(2) 541-543, (1989)) and inbacteria (Hunter et al., Biochemistry, 24, 4148-4155, (1985)). Theseorganisms can assimilate single carbon compounds, ranging in oxidationstate from methane to formate, and produce glycerol. The pathway ofcarbon assimilation can be through ribulose monophosphate, throughserine, or through xylulose-monophosphate (Gottschalk, BacterialMetabolism, Second Edition, Springer-Verlag: New York (1986)). Theribulose monophosphate pathway involves the condensation of formate withribulose-5-phosphate to form a 6 carbon sugar that becomes fructose andeventually the three carbon product glyceraldehyde-3-phosphate.Likewise, the serine pathway assimilates the one-carbon compound intothe glycolytic pathway via methylenetetrahydrofolate.

In addition to one and two carbon substrates, methylotrophic organismsare also known to utilize a number of other carbon-containing compoundssuch as methylamine, glucosamine and a variety of amino acids formetabolic activity. For example, methylotrophic yeast are known toutilize the carbon from methylamine to form trehalose or glycerol(Bellion et al., Microb. Growth C1 Compd., [Int. Symp.], 7th (1993),415-32. Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher:Intercept, Andover, UK). Similarly, various species of Candida willmetabolize alanine or oleic acid (Sulter et al., Arch. Microbiol.,153(5), 485-9 (1990)). Accordingly, the source of carbon utilized in thepresent invention may encompass a wide variety of carbon-containingsubstrates and will only be limited by the requirements of the hostorganism.

All of the above mentioned carbon substrates and mixtures thereof areexpected to be suitable in the present invention. However, preferredcarbon substrates are glycerol, dihydroxyacetone, monosaccharides,oligosaccharides, polysaccharides, and one-carbon substrates. Morepreferred are sugars such as glucose, fructose, sucrose and singlecarbon substrates such as methanol and carbon dioxide. Most preferred isglucose.

In addition to an appropriate carbon source, fermentation media mustcontain suitable minerals, salts, cofactors, buffers and othercomponents, known to those skilled in the art, suitable for the growthof the cultures and promotion of the enzymatic pathway necessary forglycerol production. Particular attention is given to Co(II) saltsand/or vitamin B₁₂ or other alternate coenzyme B 12 precursors. Forexample, E. coli and eukaryotes are unable to synthesize coenzyme B₁₂ denovo but are able to utilize coenzyme B₁₂ precursors. Preferred coenzymeB₁₂ precursors are vitamin B₁₂ and hydroxocobalamin. It is desirablethat the amount of coenzyme B₁₂ inside the host cell be approximatelyequal in molar concentration to the amount of dehydratase enzyme.

Culture Conditions

Typically, cells are grown at 30° C. in appropriate media. Preferredgrowth media in the present invention are common commercially preparedmedia such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth orYeast Malt Extract (YM) broth. Other defined or synthetic growth mediamay also be used and the appropriate medium for growth of the particularmicroorganism will be known by someone skilled in the art ofmicrobiology or fermentation science. The use of agents known tomodulate catabolite repression directly or indirectly, e.g., cyclicadenosine 3′:5′-monophosphate, may also be incorporated into thereaction media. Similarly, the use of agents known to modulate enzymaticactivities (e.g., sulphites, bisulphites and alkalis) that lead toenhancement of glycerol production may be used in conjunction with or asan alternative to genetic manipulations.

Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0,where pH 6.0 to pH 8.0 is preferred as the range for the initialcondition.

Reactions may be performed under aerobic or anaerobic conditions whereanaerobic or microaerobic conditions are preferred.

Fermentations

The present invention may be practiced using either batch, fed-batch, orcontinuous processes and any known mode of fermentation would besuitable. Additionally, cells may be immobilized on a substrate as wholecell catalysts and subjected to fermentation conditions for1,3-propanediol production.

The present process is exemplified herein as a batch method offermentation. A classical batch fermentation is a closed system wherethe composition of the media is set at the beginning of the fermentationand not artificially altered during the fermentation. Thus, at thebeginning of the fermentation the media is inoculated with the desiredorganism or organisms and fermentation is permitted to occur addingnothing to the system. Typically, however, a batch fermentation is“batch” with respect to the addition of the carbon source and attemptsare often made at controlling factors such as pH and oxygenconcentration. The metabolite and biomass compositions of the batchsystem change constantly up to the time the fermentation is stopped.Within batch cultures cells moderate through a static lag phase to ahigh growth log phase and finally to a stationary phase where growthrate is diminished or halted. If untreated, cells in the stationaryphase will eventually die. Cells in log phase generally are responsiblefor the bulk of production of end product or intermediate.

A variation on the standard batch system is the Fed-Batch fermentationsystem which is also suitable in the present invention. In thisvariation of a typical batch system, the substrate is added inincrements as the fermentation progresses. Fed-Batch systems are usefulwhen catabolite repression is apt to inhibit the metabolism of the cellsand where it is desirable to have limited amounts of substrate in themedia. Measuring the actual substrate concentration in Fed-Batch systemsis difficult and is therefore estimated on the basis of the changes ofmeasurable factors such as pH, dissolved oxygen, and the partialpressure of waste gases such as CO₂. Batch and Fed-Batch fermentationsare common and well known in the art and examples may be found in Brock,infra.

The method also is expected to be adaptable to continuous fermentationmethods. Continuous fermentation is an open system where a definedfermentation media is added continuously to a bioreactor and an equalamount of conditioned media is removed simultaneously for processing.Continuous fermentation generally maintains the cultures at a constanthigh density where cells are primarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or anynumber of factors that affect cell growth or end product concentration.For example, one method will maintain a limiting nutrient such as thecarbon source or nitrogen level at a fixed rate and allow all otherparameters to moderate. In other systems a number of factors affectinggrowth can be altered continuously while the cell concentration,measured by media turbidity, is kept constant. Continuous systems striveto maintain steady state growth conditions and thus the cell loss due tomedia being drawn off must be balanced against the cell growth rate inthe fermentation. Methods of modulating nutrients and growth factors forcontinuous fermentation processes as well as techniques for maximizingthe rate of product formation are well known in the art of industrialmicrobiology. A variety of methods are detailed by Brock, infra.

Identification and Purification of 1,3-Propanediol:

Methods for the purification of 1,3-propanediol from fermentation mediaare known in the art. For example, propanediols can be obtained fromcell media by subjecting the reaction mixture to extraction with anorganic solvent, distillation, and column chromatography (U.S. Pat. No.5,356,812). A particularly good organic solvent for this process iscyclohexane (U.S. Pat. No. 5,008,473).

1,3-Propanediol may be identified directly by submitting the media tohigh pressure liquid chromatography (HPLC) analysis. The preferredmethod is analysis of the fermentation media on an analytical ionexchange column using a mobile phase of 0.01 N sulfuric acid in anisocratic fashion.

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

EXAMPLES

General Methods

Procedures for phosphorylations, ligations and transformations are wellknown in the art. Techniques suitable for use in the following examplesmay be found in Sambrook, J. et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989).

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, eds), American Society for Microbiology, Washington,D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, Second Edition, Sinauer Associates, Inc.,Sunderland, Mass. (1989). All reagents and materials used for the growthand maintenance of bacterial cells were obtained from Aldrich Chemicals(Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL(Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.) unlessotherwise specified.

The meaning of abbreviations is as follows: “h” means hour(s), “min”means minute(s), “sec” means second(s), “d” means day(s), “mL” meansmilliliters, “L” means liters.

Cells

E. coli strain DH5α was purchased from Gibco/BRL, Gaithersburg, Md. K.pneumoniae strain ATCC 25955, K. oxytoca strain ATCC 8724, and S.typhimurium strain ATCC 23564 were purchased from the American TypeCulture Collection (ATCC), Rockville, Md. E. coli strain FM5 (ATCC53911), Amgen patent U.S. Pat. No. 5,494,816, is available from ATCC E.coli strain RK6726 (Lundrigan et al., Mol. Gen. Genet. 206, 401-407(1987)) was a gift from R. Kadner. E. coli strain CAG18491 was purchasedfrom the E. coli Genetic Stock Center, Yale University, New Haven, Conn.K. oxytoca strain M5a1 was purchased from National Collections ofIndustrial and Marine Bacteria, Ltd., Aberdeen, Scotland (NCIMB #12204).S. cerevisiae strain YPH499 (ura3-52 lys2-801 ade2-101 trp1-del63his3-del200 leu2-del1) was purchased from Stratagene, La Jolla, Calif.P. pastoris strain GTS115 (his4) was obtained from Phillips Petroleum,Bartlesville, Okla. A. niger strain FS1 is a proprietary strain fromGenencor International, Inc.

Isolation and Identification 1,3-propanediol

The conversion of glycerol to 1,3-propanediol was monitored by HPLC.Analyses were performed using standard techniques and materialsavailable to one skilled in the art of chromatography. One suitablemethod utilized a Waters Maxima 820 HPLC system using UV (210 nm) and RIdetection. Samples were injected onto a Shodex SH-1011 column (8 mm×300mm, purchased from Waters, Milford, Mass.) equipped with a ShodexSH-1011P precolumn (6 mm×50 mm), temperature controlled at 50° C., using0.01 N H₂SO₄ as mobile phase at a flow rate of 0.5 mL/min. Whenquantitative analysis was desired, samples were prepared with a knownamount of trimethylacetic acid as external standard. Typically, theretention times of glycerol (RI detection), 1,3-propanediol (RIdetection), and trimethylacetic acid (UV and RI detection) were 20.67min, 26.08 min, and 35.03 min, respectively.

Production of 1,3-propanediol was confirmed by GC/MS. Analyses wereperformed using standard techniques and materials available to one ofskill in the art of GC/MS. One suitable method utilized a HewlettPackard 5890 Series II gas chromatograph coupled to a Hewlett Packard5971 Series mass selective detector (EI) and a HP-INNOWax column (30 mlength, 0.25 mm i.d., 0.25 micron film thickness). The retention timeand mass spectrum of 1,3-propanediol generated were compared to that ofauthentic 1,3-propanediol (m/e: 57, 58).

An alternative method for GC/MS involved derivatization of the sample.To 1.0 mL of sample (e.g., culture supernatant) was added 30 uL ofconcentrated (70% v/v) perchloric acid. After mixing, the sample wasfrozen and lyophilized. A 1:1 mixture ofbis(trimethylsilyl)trifluoroacetamide:pyridine (300 uL) was added to thelyophilized material, mixed vigorously and placed at 65° C. for one h.The sample was clarified of insoluble material by centrifugation. Theresulting liquid partitioned into two phases, the upper of which wasused for analysis. The sample was chromatographed on a DB-5 column (48m, 0.25 mm I.D., 0.25 um film thickness; from J&W Scientific) and theretention time and mass spectrum of the 1,3-propanediol derivativeobtained from culture supernatants were compared to that obtained fromauthentic standards. The mass spectrum of TMS-derivatized1,3-propanediol contains the characteristic ions of 205, 177, 130 and115 AMU.

Assay for cob(I)alamin Adenosyltransferase and Cob(II)Alamin ReductaseActivity.

Cob(I)alamin adenosyltransferase may be assayed as described by S.-J.Suh and J. C. Escalante-Semerena, J. Bacteriol. 177, 921-925 (1995) orL. Debussche et al., J. Bacteriol. 173, 6300-6302 (1991). Alternatively,cob(I)alamin adenosyltransferase may be determine by an in vivo assay asdescribed in Example 1.

Cob(II)alamin reductase may be assayed as described by L. Debussche etal., J. Bacteriol. 174, 7452-7454 (1992).

Isolation and Cloning of Genes Encoding Glycerol Dehydratase (Dhab) and1,3-propanediol Oxidoreductase (dhaT)

Identification and isolation of dhaB and dhaT were done essentially asdescribed in U.S. Pat. No. 5,686,276 and those methods are herebyincorporated by reference. Cosmid vectors and cosmid transformationmethods were used to clone large segments of genomic DNA from bacterialgenera known to possess genes capable of processing glycerol to1,3-propanediol. Specifically, genomic DNA from K. pneumoniae ATCC 25955was isolated by methods well known in the art and digested with therestriction enzyme Sau3A for insertion into a cosmid vector Supercos 1and packaged using GigapackII packaging extracts. Following constructionof the vector E. coli XL1-Blue MR cells were transformed with the cosmidDNA. Transformants were screened for the ability to convert glycerol to1,3-propanediol by growing the cells in the presence of glycerol andanalyzing the media for 1,3-propanediol formation.

Two of the 1,3-propanediol positive transformants were analyzed and thecosmids were named pKP1 and pKP2. DNA sequencing revealed extensivehomology to the glycerol dehydratase gene (dhaB) from C. freundii,demonstrating that these transformants contained DNA encoding theglycerol dehydratase gene.

A 12.1 kb EcoRI-SalI fragment from pKP1, subcloned into pIB131 (IBIBiosystem, New Haven, Conn.), was sequenced and termed pHK28-26 (SEQ IDNO:10). Sequencing revealed the loci of the relevant open reading framesof the dha operon encoding glycerol dehydratase and genes necessary forregulation. Referring to SEQ ID NO:10, a fragment of the open readingframe for dhaK (encoding dihydroxyacetone kinase) is found at bases1-399; the open reading frame dhaD (encoding glycerol dehydrogenase) isfound at bases 983-2107; the open reading frame dhaR (encoding therepressor) is found at bases 2209-4134; the open reading framedhaT(encoding 1,3-propanediol oxidoreductase) is found at bases5017-6180; the open reading frame dhaB1 (encoding the alpha subunitglycerol dehydratase) is found at bases 7044-8711; the open readingframe dhaB2 (encoding the beta subunit glycerol dehydratase) is found atbases 8724-9308; the open reading frame dhaB3 (encoding the gammasubunit glycerol dehydratase is found at bases 9311-9736; and the openreading frame dhaBX (encoding a protein of unknown function) is found atbases 9749-11572. Additionally, the open reading frame orfY (encoding aprotein of unknown function) is found at bases 6202-6630; the openreading frame orfX (encoding a protein of unknown function) is found atbases 4643-49, and the open reading frame orfW (encoding a protein ofunknown function) is found at bases 4112-4642.

Construction of General Purpose Expression Plasmids for Use inTransformation of Escherichia coli

Construction of Expression Vector pTacIQ

The E. coli expression vector pTacIQ was prepared by inserting lacIqgene (Farabaugh, P. J., Nature 274 (5673) 765-769, (1978)) and tacpromoter (Amann et al., Gene 25, 167-178 (1983)) into the restrictionendonuclease site EcoRI of pBR322 (Sutcliffe, Cold Spring Harb. Symp.Quant. Biol. 43, 77-90 (1979)). A multiple cloning site and terminatorsequence (SEQ ID NO:11) replaces the pBR322 sequence from EcoRI toSphiI.

Subcloning the Glycerol Dehydratase Genes (dhaB1,2,3, X)

The open reading frame for the dhaB3 gene was amplified from pHK 28-26by PCR using primers (SEQ ID NO:12 and SEQ ID NO:13) incorporating anEcoRI site at the 5′ end and a XbaI site at the 3′ end. The product wassubcloned into pLitmus29 (New England Biolab, Inc., Beverly, Mass.) togenerate the plasmid pDHAB3 containing dhaB3.

The region containing the entire coding region for dhaB1, dhaB2, dhaB3and dhaBX of the dhaB operon from pHK28-26 was cloned intopBluescriptIIKS+ (Stratagene, La Jolla, Calif.) using the restrictionenzymes KpnI and EcoRI to create the plasmid pM7.

The dhaBX gene was removed by digesting plasmid pM7 with ApaI and XbaI,purifying the 5.9 kb fragment and ligating it with the 325-bp ApaI-XbaIfragment from plasmid pDHAB3 to create pM11 containing dhaB1, dhaB2 anddhaB3.

The open reading frame for the dhaB1 gene was amplified from pHK28-26 byPCR using primers (SEQ ID NO:14 and SEQ ID NO:15) incorporating aHindIII site and a consensus ribosome binding site at the 5′ end and aXbaI site at the 3′ end. The product was subcloned into pLitmus28 (NewEngland Biolab, Inc.) to generate the plasmid pDT1 containing dhaB1.

A NotI-XbaI fragment from pM11 containing part of the dhaB1 gene, thedhaB2 gene and the dhaB3 gene was inserted into pDT1 to create the dhaBexpression plasmid, pDT2. The HindIII-XbaI fragment containing thedhaB(1,2,3) genes from pDT2 was inserted into pTacIQ to create pDT3.

Subcloning the 1,3-propanediol Dehydrogenase Gene (dhaT)

The KpnI-SacI fragment of pHK28-26, containing the 1,3-propanedioldehydrogenase (dhaT) gene, was subcloned into pBluescriptII KS+ creatingplasmid pAH1. The dhaT gene was amplified by PCR from pAH1 as templateDNA and synthetic primers (SEQ ID NO:16 with SEQ ID NO:17) incorporatingan XbaI site at the 5′ end and a BamHI site at the 3′ end. The productwas subcloned into pCR-Script (Stratagene) at the SrfI site to generatethe plasmids pAH4 and pAH5 containing dhaT. The plasmid pAH4 containsthe dhaT gene in the right orientation for expression from the lacpromoter in pCR-Script and pAH5 contains dhaT gene in the oppositeorientation. The XbaI-BamHI fragment from pAH4 containing the dhaT genewas inserted into pTacIQ to generate plasmid pAH8. The HindIII-BamHIfragment from pAH8 containing the RBS and dhaT gene was inserted intopBluescriptIIKS+ to create pAH11.

Construction of an Expression Cassette for dhaT and dhaB(1,2,3)

An expression cassette for dhaT and dhaB(1,2,3) was assembled from theindividual dhaB(1,2,3) and dhaT subclones described previously usingstandard molecular biology methods. A SpeI-SacI fragment containing thedhaB(1,2,3) genes from pDT3 was inserted into pAH11 at the SpeI-SacIsites to create pAH24. A SalI-XbaI linker (SEQ ID NO:18 and SEQ IDNO:19) was inserted into pAH5 which was digested with the restrictionenzymes SalI-XbaI to create pDT16. The linker destroys the XbaI site.The 1 kb SalI-MluI fragment from pDT16 was then inserted into pAH24replacing the existing SalI-MluI fragment to create pDT18. pDT21 wasconstructed by inserting the SalI-NotI fragment from pDT18 and theNotI-XbaI fragment from pM7 into pCL1920 (SEQ ID NO:20). The glucoseisomerase promoter sequence from Streptomyces (SEQ ID NO:21) was clonedby PCR and inserted into EcoRI-HinDIII sites of pLitmus28 to constructpDT5. pCL1925 was constructed by inserting EcoRI-PvuII fragment of pDT5into the EcoRI-PvuI site of pCL1920. pDT24 was constructed by cloningthe HinDIII-MluII fragment of pDT21 and the MluI-XbaI fragment of pDT21into the HinDIII-XbaI sites of pCL1925.

Example 1

btuR and cobA: Gene Isolation Plasmid Construction and Activity

The E. coli DH5α (deoR endA1 gyrA96 hsdR17(rk− mk+) recA1 relA11 supE44thi-1 Δ(lacZYA-argFV169)) strain used for isolation of the cob(I)alaminadenosyltransferase (btuR) gene was purchased from Gibco BRL(Gaithersburg, Md.). btuR was amplified from chromosomal DNA by PCRusing synthetic primers (SEQ ID NO:22 with SEQ ID NO:23) incorporating aribosome binding site flanked by HindIII and BamHI sites at the 5′ endand a PstI site at the 3′ end. The product was subcloned into pCR-Script(Stratagene, Madison, Wis.) at the SrfI site to generate the plasmidpAH61 containing btuR in the correct orientation for expression from thelac promoter.

The activity of pAH61 was demonstrated by restoring 1,3-propanediolproduction in an E. coli, btuR minus background (RK6726, Lundrigan etal., Mol. Gen. Genet. 206, 401-407 (1987)). E. coli strains RK6726/pDT24and RK6726/pDT24/pAH61 were grown in erlenmeyer flasks containing mediumat one fifth the volume of flask capacity. The plasmid pDT24 containsthe genes encoding glycerol dehydratase and 1,3-propanedioloxidoreductase). Medium, titrated to pH 6.8 with HCl, contained 0.2 MK₂HPO₄, 2.0 g/L citric acid, 2.0 g/L MgSO₄.7H₂O, 1.2 mL 98% H₂SO₄, 0.30g/L ferric ammonium citrate, 0.20 g/L CaCl₂2H₂O, 5 g/L yeast extract, 15g/L D-glucose, 60 g/L glycerol, 5 mL per liter of Modified Balch/EsTrace-Element Solution (Cote, R. J., and Gherna, R. L. In Methods forGeneral and Molecular Bacteriology; Gerhardt, P. et al., Eds; AmericanSociety for Microbiology: Washington, D.C., 1994; p. 158), and 50 mg/Lvitamin B₁₂. In addition, 50 ug/mL spectinomycin and 100 ug/mLcarbencillin were used to maintain the plasmids pDT24 and pAH61,respectively. The shake flasks were incubated at 33° C. with vigorousshaking. After 48 hours, RK6726/pDT24/pAH61 (OD₆₀₀=9.8 AU) produced 4.0g/L 1,3-propanediol. In the control experiment with RK6726/pDT24(OD₆₀₀=10.1 AU), 1,3-propanediol was not detected.

The S. typhimurium strain (ATCC 23564) used for isolation of thecob(I)alamin adenosyltransferase (cobA) gene was purchased from theAmerican Type Culture Collection (Rockville, Md.). cobA was amplifiedfrom chromosomal DNA by PCR using synthetic primers (SEQ ID NO:24 withSEQ ID NO:25) incorporating a ribosome binding site flanked by HindIIIand BamHI sites at the 5′ end and a PstI site at the 3′ end. The productwas subcloned into pCR-Script (Stratagene, Madison, Wis.) at the SrfIsite to generate the plasmid pAH63 containing cobA in the correctorientation for expression from the lac promoter. The activity of pAH63was demonstrated as described for pAH61.

Example 2

Cob(II)alamin Reductase: Gene Isolation and Plasmid Construction Media

Synthetic S12 medium is used in the screening of bacterial transformantsfor the ability to grow in the absence of methionine in the presence ofeither vitamin B₁₂ or coenzyme B₁₂. S12 medium contains: 10 mM ammoniumsulfate, 50 mM potassium phosphate buffer, pH 7.0, 2 mM MgCl₂, 0.7 mMCaCl₂, 50 uM MnCl₂, 1 uM FeCl₃, 1 uM ZnCl, 1.7 uM CuSO₄, 2.5 uM CaCl₂,2.4 uM Na₂MoO₄, and 2 uM thiamine hydrochloride. S12 medium issupplemented with 0.2% D-glucose, 2 mg/L uracil, and 400 ug/L vitaminB₁₂ or coenzyme B₁₂.

Selection for a cob(II)alamin Reductase Defective Strain

E. coli strain CAG18491 (F⁻, λ⁻, rph-1, metE3079::Tn10) was purchasedfrom the E. coli Genetic Stock Center, Yale University (New Haven,Conn.). All incubations are at 37° C. CAG18491 is grown in LB mediumcontaining 10 mg/L tetracycline to an A₆₀₀ 0.3-0.4 AU, made competent byCaCl₂ treatment, and transformed with the btuR plasmid pAH61.Transformants are selected following overnight growth on LB platescontaining 10 mg/L tetracycline and 50 mg/L ampicillin. A singletransformant is grown in LB containing 10 mg/L tetracycline and 50 mg/Lampicillin to an A₆₀₀ 0.5-0.6 AU. 1 M MgSO₄ is added to give a finalconcentration of 10 mM, and bacteriophage λ::Tn5 is added to give anm.o.i. of 1. After 10 min to allow phage infection the culture isallowed to grow for an additional 60 min. Serial dilutions are plated onLB containing 10 mg/L tetracycline, 50 mg/L ampicillin and 25 mg/Lkanamycin. Following overnight growth, several thousand colonies arepooled by scraping plates bearing well-separated colonies. The pool ofcells is washed extensively by centrifugation and resuspension in S12medium. Serial dilutions are plated on S12 plates containing coenzymeB₁₂ and incubated in the dark for 2 days. Colonies are replica-platedonto S12 plates containing vitamin B₁₂ and 0.1 mMisopropyl-β-D-galactopyranoside (IPTG) and incubated in the dark for 2days. Colonies that fail to grow on vitamin B₁₂ but grow on coenzyme B₁₂are enriched for mutations in the vitamin B₁₂ to coenzyme B₁₂conversion, but will not be mutated in btuR because of the presence ofthe multicopy btuR plasmid pAH61, nor will they be defective incobalamin transport or methionine synthesis because they grow on minimalmedia containing coenzyme B₁₂. Putative cobalt reductase mutants areassayed for the loss of cob(II)alamin reductase using the in vivo assaywith cells treated with toluene and incubated with reducedhydroxycobalamin.

A characterized cob(II)alamin reductase mutant is used to select bycomplementation a gene that will restore growth on S12 plates containingvitamin B₁₂ and IPTG. Chromosomal DNA is isolated from CAG18491, or fromother strains or species that are metE, and partially digested with therestriction enzyme Sau3A. The Sau3A fragments are ligated into the BamHIsite of plasmid pACYC184 and transformed into the cob(II)alaminreductase minus strain. Plasmid pACYC184 (ch1R) is compatible with pAH61(ampR), and the presence of Tn5 (kanR) and Tn10 (tetR) are confirmed byresistance to the appropriate antibiotic. The transformedlibrary-containing cells are plated on S12 plates containing vitamin B₁₂and 0.1 mM IPTG and incubated in the dark for 2 days. Plasmids thatrestore growth of cells by complementation under these growth conditionsare screened using the in vitro cob(II)alamin reductase assay to confirmthe presence of the cloned gene.

Example 3 Host Construction

E. coli strain FM5 is co-transformed with the dha plasmid pDT24 (specR),the btuR plasmid pAH61 (ampR), and the cob(II)alamin reductase plasmidbased on pACYC184 (ch1R). Selection is on LB plates containing 50 mg/Lspectinomycin, 50 mg/L ampicillin and 100 mg/L chloramphenicol. Coloniesresistant to all three antibiotics are used for 1.3-propanediolproduction.

Example 4 Enhanced 1,3-propanediol Production

Growth of cells from Example 3 to give an improvement in 1,3-propanediolproduction is carried at 37° C. in shake-flask cultures (erlenmeyerflasks, liquid volume one tenth that of total volume).

E. coli strain FM5/pDT24 cotransformed with pAH61 and the cob(II)alaminreductase plasmid is grown in 250 mL flasks containing 25 mL of mediumat 30° C. with shaking at 250 rpm. FM5/pDT24 (the parent strain) isgrown in parallel as the control. Medium, titrated to pH 6.8 with NH₄OH,contains 0.2 M KH₂PO₄, 2.0 g/L citric acid, 2.0 g/L MgSO₄.7H₂O, 1.2 mL98% H₂SO₄, 0.30 g/L ferric ammonium citrate, 0.20 g/L CaCl₂.2H₂O, 5 mLof trace metal mix, 5 g/L yeast extract, 10 g/L D-glucose, 30 g/Lglycerol and 5 mg/L of either vitamin B₁₂ or hydroxocobalamin. Tracemetal mix contains (g/L): Na₂SO₄ (4.0), MnSO₄ H₂O (0.80), ZnSO₄ 7H₂O(1.6), CoSO₄ (0.52), CuSO₄ 5H₂O (0.12), and FeSO₄ 7H₂O (4.0). Inaddition, the appropriate antibiotics are present in order to maintainplasmid stability.

Flasks are inoculated to an initial OD600 of approximately 0.01 AU, pHis maintained above pH 6.2 with the addition of 0.5 N KOH, and theglucose concentration is maintained above 2 g/L with the addition of a50% (w/w) solution. pH is monitored using ColorpHast strips (EM Science,Gibbstown, N.J.). Glucose concentration is monitored using the Trinderenzymatic assay (Sigma, St. Louis, Mo.). At various times, aliquots areremoved in order to determine 1,3-propanediol concentration (by gc orhplc analysis as described above) and cell density (OD₆₀₀). The strainFM5/pDT24 cotransformed with pAH61 and the cob(II)alamin reductaseplasmid shows increased 1,3-propanediol production compared to theparent strain.

1. A process for the bio-production of 1,3-propanediol comprising: (i)contacting a transformed host cell with at least one fermentable carbonsource and an effective amount of coenzyme B₁₂ precursor whereby1,3-propanediol is produced, the transformed host cell comprising: (a)at least one copy of genes encoding a protein having a glyceroldehydratase activity or a diol dehydratase activity selected from thegroup consisting of Klebsiella pneumoniae dhaB1-3 as shown in SEQ IDNO:4, SEQ ID NO:5, and SEQ ID NO:6; (b) at least one copy of a geneencoding a protein having a 1,3-propanediol oxidoreductase activity ofKlebsiella pneumoniae dhaT as shown in SEQ ID NO:7; and (c) one copy ofa gene encoding a protein having a cob(I)alamin adenosyltransferaseactivity selected from the group consisting of Escherichia coil btuR asshown in SEQ ID NO:1, Salmonella typhimurium cobA as shown in SEQ IDNO:2, and Pseudomonas denitrificans cobO as shown in SEQ ID NO:3;wherein the genes of (i)(a)-(i)(c) are heterologous to the host cell andexpress their respective gene products in active form, and (ii)recovering the 1,3-propanediol produced from step (i).
 2. The processaccording to claim 1 wherein the fermentable carbon source is selectedfrom the group consisting of fermentable carbohydrates, single-carbonsubstrates, and mixtures thereof.
 3. The process according to claim 1wherein the fermentable carbon source is selected from the groupconsisting of monosaccharides, oligosaccharides, polysaccharides, singlecarbon substrates, glycerol, dihydroxyacetone and carbon-containingamines.
 4. The process according to claim 1 wherein the transformed hostcell is selected from the group consisting of bacteria, yeast, andfilamentous fungi.
 5. The process according to claim 4 wherein thetransformed host cell is selected from the group consisting ofCitrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter,Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces,Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula,Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella,Bacillus, Streptomyces, and Pseudomonas.
 6. An improved process for thebio-production of 1,3-propanediol comprising: (i) contacting atransformed host cell with at least one carbon source selected from thegroup consisting of fermentable carbohydrates, single-carbon substratesand mixtures thereof, and an effective amount of coenzyme B₁₂ precursor,whereby 1,3-propanediol is produced; the transformed host cellcomprising: (a) at least one copy of genes encoding a protein having aglycerol dehydratase activity or a diol dehydratase activity selectedfrom the group consisting of Klebsiella pneumoniae dhaB1-3 as shown inSEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; (b) at least two copies of agene encoding a protein having a 1,3-propanediol oxidoreductase activityof Klebsiella pneumoniae dhaT as shown in SEQ ID NO:7; and (c) at leastone copy of a gene encoding a protein having a cob(I)alaminadenosyltransferase activity selected from the group consisting ofEscherichia coil btuR as shown in SEQ ID NO:1, Salmonella typhimuriumcobA as shown in SEQ ID NO:2, and Pseudomonas denitrificans cobO asshown in SEQ ID NO:3; wherein the genes of (i)(a)-(i)(c) areheterologous and express their respective gene products in active form;and (ii) recovering the 1,3-propanediol produced from step (i); theimprovement comprising an increase in the production of 1,3-propanediolas compared with a bio-process wherein the gene of (i)(c) is not presentin multicopy in the transformed host cell.
 7. A process for regulatingthe bio-production of 1,3-propanediol comprising: (i) contacting atransformed host cell with (a) at least one carbon source selected fromthe group consisting of fermentable carbohydrates, single-carbonsubstrates and mixtures thereof and (b) an effective amount of coenzymeB₁₂ precursor whereby 1,3-propanediol is produced, the transformed hostcell comprising: (a)at least one copy of genes encoding a protein havinga glycerol dehydratase activity or a diol dehydratase activity selectedfrom the group consisting of Klebsiella pneumoniae dhaB1-3 as shown inSEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; (b) at least one copy of agene encoding a protein having a 1,3-propanediol oxidoreductase activityof Klebsiella pneumoniae dhaT as shown in SEQ ID NO:7; and (c) at leastone copy of a gene encoding a protein having a cob(I)alaminadenosyltransferase activity selected from the group consisting ofEscherichia coil btuR as shown in SEQ ID NO:1, Salmonella typhimuriumcobA as shown in SEQ ID NO:2, and Pseudomonas denitrificans cobO asshown in SEQ ID NO:3; wherein the gene of (i)(c) is selectivelyinhibited whereby the metabolism of coenzyme B₁₂ precursor is regulated.8. The process of any one of claims 1, 6 or 7 wherein the effectiveamount of coenzyme B₁₂ precursor is produced de novo by the transformedhost cell.
 9. The process of any one of claims 1, 6 or 7 wherein theeffective amount of coenzyme B₁₂ precursor is at a 0.1- to 10.0-foldmolar ratio to the amount of dehydratase present.